Note: Descriptions are shown in the official language in which they were submitted.
WO 92/18525 PC'T/US92/01978
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METHOD OF SEPARATING A PEPTIDE FROM A RESIN
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to the field of
separating a peptide from a resin to which the peptide
is bonded, following solid phase peptide synthesis.
Description of the Background Art
Solid phase peptide synthesis involves assembling
amino acids into a peptide of any desired sequence
while one end of the chain is anchored to an insoluble
support. The insoluble support is a synthetic polymer
which bears reactive groups. The amino acid which
forms the C-terminal reside of the peptide to be
synthesized is converted to a derivative in which its
amino group is protected by a labile protecting group.
The derivative of the C-terminal amino acid is coupled
to the reactive polymer. A reagent is applied to the
protected aminoacyl polymer to remove the labile
blocking group from the amino acid residue. The
reagent must not harm the link of the C-terminal
residue to the polymer in any way. Moreover, if the
amino acid attached to the polymer contains a side-
chain reactive functional group, that functional group
must be blocked by a stable blocking group which will
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remain completely intact throughout the synthesis, but
which can be removed finally to yield the free peptide.
Following removal of the labile protecting group, the
next amino acid is coupled to the aminoacyl polymer by
use of a suitable coupling reaction. Again, the a-amino
group must be protected with a labile protecting group.
This cycle of deprotection and coupling is then repeated
with each amino acid which is to be incorporated into the
peptide chain. Finally, after the entire blocked peptide
has been assembled on the polymer support, a different
type of reagent is applied to cleave the peptide from the
polymer and allow it to be dissolved. The blocking
groups which have protected side-chain functional groups
must also be removed, and usually are chosen so that they
can be removed simultaneously with cleavage of the
peptide from the resin.
One reagent for cleavage of peptide from the resin
at the end of the synthesis is anhydrous liquid hydrogen
fluoride (HF). HF cleavage is generally done at 0°C for
30 minutes. Such conditions will generally cleave the
peptide effectively from the resin and remove all side-
chain blockings groups. The HF then is removed, e.g.,
under vacuum, and the cleaved peptide then is separated
from the resin, see, e.g., Nomizu et al., Int. J. Peptide
Protein Res 37:145-152 (1991).
EP-A-0 331 073 describes partial peptide cleavage
with trifluoroacetic acid (TFA) at a temperature much
SUBSTITUTE SHEET
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higher than with HF (28°C), followed by removal of resin
and evaporation of the TFA.
30 A common problem associated with HF cleavage of
resin-peptide following solid phase peptide synthesis is
side reactions caused by prolonged contact of the peptide
with HF. In order to avoid "bumping", or a sudden
surge of HF/resin slurry, the process is carried out
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WO 92/18525 PCT/US92/01978
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peptide to HF and causing the above-noted side
reactions. The problem is even more pronounced in a
large scale cleavage, e.g., greater than one liter,
when a large quantity of HF cannot rapidly be removed
after a proper reaction time has elapsed. Furthermore,
constant monitoring and adjustment of vacuum level are
required to control the process.
After removal of HF, the peptide is extracted from
the resin with appropriate solvent(s). However, the
low solubility of some peptides makes it difficult to
completely recover the peptide from the resin,
resulting in a lower yield.
There thus remains a need in the art for improved
methods of separating peptides from resins so as to
reduce or eliminate side reactions..
SUMMARY OF THE INVENTION
In accordance with the present invention, a method
of separating a peptide from a resin to which the
peptide is bound comprises treating the resin-bound
peptide with hydrogen fluoride so as to cleave the
peptide from the resin and form a first mixture of
peptide, hydrogen fluoride and resin. The resin is
removed from the first mixture so as to provide a
resin-free second mixture including the polypeptide and
the hydrogen fluoride. The polypeptide of the second
mixture then is separated from the hydrogen fluoride
present therein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is applicable to the well-
known technique of solid phase peptide synthesis such
as is described in Steward and Young, The Chemistry of
WO 92/18525 PCT/US92/01978
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Solid Phase Peptide Synthesis, Pierce Chemical Company
(1984).
Peptides are formed in solid phase peptide
synthesis on a resin support, such as 1~ cross-linked
polystyrene (copolymer of styrene with 1~
divinylbenzene), MBHA, BHA, Pam resins and the like.
Construction of the peptide is initiated by attaching
to the resin a first amino acid which will form the C-
terminal residue of the peptide to be synthesized. For
example, the polystyrene resin beads can be
functionalized by chloromethylation, which introduces
benzyl chloride-type groups into the polymer. These
halogens are reactive, and when treated with the salt
of a protected amino acid will form an ester, linking
the protected amino acid covalently to the resin as a
substituted benzyl ester.
Typically, the oc-amino function of the amino acid
is protected by a Boc group. After bonding of the
initial amino acid to the resin, the Boc protecting
group is removed by treatment with, for example, dilute
solutions of strong acids such as 25$ trifluoroacetic
acid (TFA) in dichloromethane (DCM). After removal of
the Boc protecting group, the newly exposed amino group
is present as an acid salt which can be converted to a
free base by treatment with a tertiary amine, such as
triethyl amine in DCM.
The peptide is then "grown" by sequentially
coupling Boc protected amino acids to the aminoacyl
resin, typically by use of dicyclohexylcarbodiimide
(DCC), with each additional amino acid being added
after removal of the Boc-group from the last amino acid
WO 92/18525 ~ ~ ~ ~ ~ ~ ~ PCT/US92/01978
in the chain. Following completion of assembly of the
desired blocked peptide on the resin, the resin-bonded
peptide can be treated with anhydrous HF to cleave the
linkage of the peptide to the resin in order to
5 liberate the free peptide.
In accordance with one embodiment of the present
invention, the resin-bonded peptide is treated with
hydrogen fluoride at a cleavage temperature of from
about -5°C to about 5°C for from about one-half hour to
about one hour so as to cleave the peptide from the
resin with the peptide substantially intact.
After cleaving the peptide from the resin and
thereby forming a mixture containing resin, cleaved
peptide and hydrogen fluoride, the mixture is quickly
filtered to remove the resin, leaving HF and peptide
remaining in the filtrate.
Since peptides are soluble in HF, the majority of
the cleaved peptide remains in the filtrate, and can be
separated from the HF by, for example, vacuum
distillation of the HF from the filtrate.
After removal of the HF from the filtrate, the
peptide present in the residue can be washed with a
solvent in which HF is soluble and the peptide is
insoluble, so as to remove residual HF from the
distillation residue. Suitable solvents for washing
the peptide include diethyl ether and ethyl acetate.
Any cleaved peptide remaining with the resin
subsequent to filtration and vacuum distillation can be
separated from the resin by repeating the HF extraction
~ as described above, or by methods well known in the
art, such as by extraction with acetic acid.
WO 92/18525 PCT/US92/01978
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The invention is further illustrated by the
following examples, which are not intended to be
limiting.
EXAMPLE I
A resin-bonded tetrapeptide (leu-Ala-Gly-Val-Pam-
resin) was cleaved with HF at 0°C for one-half hour in
a teflon reactor. At the end of the cleavage, the
peptide/resin/HF slurry was filtered through a pre-
chilled teflon filter into a container. The HF
filtrate was evaporated under vacuum, and after 15
minutes, a faint, white film was found deposited on the
container wall. This film was extracted with ethyl
acetate (EtOAc)/1 M acetic acid (HOAc) (15 ml each).
The aqueous phase was collected from a separatory
funnel and lyophilized. The resin which had been
removed from the filtrate was washed with EtOAc and
extracted with HOAc. Crude peptide present in the
resin-derived extract was lyophilized. The total crude
peptide recovered from both the filtrate and the resin
was 148.4 milligrams per gram peptide resin, with 96~
recovered from the HF filtrate and 4~ from the resin.
EXAMPLE II
Resin bound Desmopressin-MBHA (20.6 gm) was
swollen with 20 ml anisole and brought to contact with
anhydrous HF (200 ml) at 0°C for one hour. The
peptide/HF solution was filtered into a Teflon
evaporator. Another 100 ml HF was charged into
cleavage reactor and filtered into the same Teflon
evaporator. HF was removed by vacuum distillation. No
bumping was observed during the distillation even
though a full vacuum was applied. At the end of the
PCT/US92/01978
WO 92/18525
7
distillation, a sticky, light brown mass of crude
peptide was obtained. After repeated washing with
ethyl ether, the crude peptide became less sticky and
the color turned to light yellowish. The mass was
estimated to be 12-14 gram. The resin, after being
washed with ethyl ether, was extracted with 1 M acetic
acid solution. No measurable quantity of peptide,
however, was obtained from the resin which suggest that
the peptide had been completely extracted with HF.
The prior art problems of "bumping" and prolonged
exposure of peptide to HF causing undesired side
reactions are eliminated by separating HF from resin
after the cleavage reaction is completed. The
separation of HF and resin can be achieved by
filtration, centrifugation or any other means of
separating liquid from solid. The HF then can be
removed by vacuum distillation in the absence of resin
beads. Without being bound to any particular theory,
it is believed that with the present invention, where
the solid particles are very small in the slurry and
the consistency is very high, the slurry displays
properties, such as viscosity, that are quite different
from those of true liquids. It is this non-Newtonian
behavior that is believed to cause the "bumping"
problem in prior art HF removal from resin beads.
By removing resin prior to separating HF from
peptide in accordance with the present invention,
several significant advantages are achieved. A high
vacuum can be applied for separation of HF without
causing any "bumping" problem. HF is removed at a
higher rate, thus reducing the contact time between HF
WO 92/18525 PCT/US92/01978
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and peptide. Because there is no "bumping" problem, a
large scale process is easy to operate.
Additionally, HF is one of the best solvents for
peptide. Therefore, by using HF both as a cleavage
reagent and as an extractant for peptide, the yield for
some "insoluble" peptides is improved.
After HF is removed from the filtrate, the crude
peptide can be effectively washed with a suitable
solvent' to remove small organic impurities. The solid
crude peptide obtained in this manner may offer an
opportunity for purification in that it may be
crystallized in an appropriate solvent. Thus, a final
HPLC purification step may be improved or even
eliminated.
Furthermore, the low-high HF cleavage method
proposed by Tam, et al., Peptide Research 1:6 (1988),
can be greatly improved by utilization of the present
invention, if the solid polymer support used is MBHA,
BHA or Pam resin. The low-high HF scheme proposed by
Tam, et al. calls for a vacuum distillation of low-HF
(DMS/HF/scavenger) which may take hours to accomplish,
before proceeding to the high-HF (scavenger/HF) step.
By utilization of the present invention, the HF under
low-HF conditions can be easily filtered off from the
reaction vessel and additional amounts of HF can be
charged into the reactor to effect the final cleavage
of peptide from resin under high-HF conditions.
The present invention minimizes contact time
between the peptide and the HF, and HF remaining after
vacuum distillation is diluted by the charged solvent
so as to further reduce side-reactions. If desired,
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WO 92/18525 PCT/US92/01978
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the peptides can be repeatedly washed before being
dried and no lyophilization is needed to obtain dry
peptide powder. The peptide powder can easily be
stored in a freezer, and the peptide in dry powder form
allows better process control of subsequent
purification steps.
The present invention provides an effective method
for reducing the side reaction problems associated with
HF cleavage of resin-bonded peptides. Since many
modifications, variations and changes in detail may be
made to the described embodiments, it is intended that
all matter in the foregoing description be interpreted
as illustrative and not in a limiting sense.
